Type 2 diabetes mellitus, or “late onset” diabetes, is a common, degenerative disease affecting 5 to 10 percent of the population in developed countries. The propensity for developing type 2 diabetes mellitus (“type 2 DM”) is reportedly maternally inherited, suggesting a mitochondrial genetic involvement. (Alcolado, J. C. and Alcolado, R., Br. Med. J. 302:1178-1180 (1991); Reny, S. L., International J. Epidem. 23:886-890 (1994)). Diabetes is a heterogeneous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals.
At the cellular level, the pathologic phenotype that may be characteristic of the presence of, or risk for predisposition to, late onset diabetes mellitus includes the presence of one or more indicators of altered mitochondrial respiratory function, for example impaired insulin secretion, decreased ATP synthesis and increased levels of reactive oxygen species. Studies have shown that type 2 DM may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from impaired glucose tolerance (IGT). Following a glucose load, circulating glucose concentrations in IGT patients rise to higher levels, and return to baseline levels more slowly, than in unaffected individuals. A small percentage of IGT individuals (5-10%) progress to non-insulin dependent diabetes (NIDDM) each year. This form of diabetes mellitus, type 2 DM, is associated with decreased release of insulin by pancreatic beta cells and a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, peripheral and sensory neuropathies and blindness.
Glucose-mediated insulin secretion from the pancreatic beta cell is triggered by a complex sequence of intracellular events. Glucose is taken up by the beta cell via GLUT-2 glucose transporters; it is subsequently phosphorylated by glucokinase to glucose-6-phosphate, which enters the glycolytic pathway. The reducing equivalents (NADH) and substrate (pyruvate) produced through glycolysis enter the mitochondria and fuel increased respiration and oxidative phosphorylation. The consequent rise in cellular ATP levels triggers closure of the K+-ATP channels at the plasma membrane, depolarizing the membrane and permitting influx of calcium. Calcium appears to have two main roles: stimulating release of insulin from the cells (e.g., Kennedy et al., 1996 J. Clin. Invest. 98:2524; Maechler et al., 1997 EMBO J. 16:3833), and acting as a “feed-forward” regulator of mitochondrial ATP production (e.g., Cox and Matlib, 1993 Trends Pharmacol. Sci. 14:408). The latter is accomplished by mitochondrial uptake of calcium through the mitochondrial calcium uniporter (e.g., Newgard et al., 1995 Ann. Rev. Biochem. 64:689; Magnus et al., 1998 Am. J. Physiol. 274:C1174-C1184). The rise in mitochondrial calcium stimulates respiration and oxidative phosphorylation through stimulation of calcium-sensitive dehydrogenase (Rutter et al., 1988 Biochem. J. 252:181; Rutter et al., 1993 J. Biol. Chem. 268:22385). However, the rise in mitochondrial calcium is transient, since calcium returns to the cytoplasm through regulated calcium efflux channels, for instance a mitochondrial calcium antiporter such as the mitochondrial calcium/sodium antiporter (MCA) also known as the mitochondrial sodium/calcium exchanger (mNCE; see, e.g., Newgard 1995; Magnus 1998; for a general review of mitochondrial membrane transporters, see, e.g., Zonatti et al., 1994 J. Bioenergetics Biomembr. 26:543 and references cited therein). The use of MCA inhibitors has been contemplated for their potential effects on cardiac function (e.g., Cox and Matlib, 1993 Trends Pharmacol. Sci. 14:408-413), but such use has not been suggested for certain other indications such as diabetes. Thus, for example, while elevated intramitochondrial calcium concentration has been correlated with insulin secretion and oxidative ATP synthesis, as noted above (e.g., Kennedy et al., 1996 J. Clin. Invest. 98:2524; Maechler et al., 1997 EMBO J. 16:3833; Cox and Matlib, 1993 Trends Pharmacol. Sci. 14:408), no inducer-effector relationship between oxidative ATP synthesis and insulin secretion has been universally accepted (see, e.g., Newgard, 1995 Ann. Rev. Biochem. 64:689). Moreover, currently available inhibitors of the MCA are regarded as either not specific for the MCA, or useful only at extremely high concentrations, precluding their apparent suitability for pharmaceutical compositions (Cox and Matlib, 1993 Trends Pharmacol. Sci. 14:408-413).
Current pharmacological therapies for type 2 DM include injected insulin, and oral agents that are designed to lower blood glucose levels. Currently available oral agents include: (i) the sulfonylureas, which act by enhancing the sensitivity of the pancreatic beta cell to glucose, thereby increasing insulin secretion in response to a given glucose load; (ii) the biguamides, which improve glucose disposal rates and inhibit hepatic glucose output; (iii) the thiazolidinediones, which improve peripheral insulin sensitivity through interaction with nuclear peroxisome proliferator-activated receptors (PPAR, see, e.g., Spiegelman, 1998 Diabetes 47:507-514; Schoonjans et al., 1997 Curr Opin. Lipidol. 8:159-166; Staels et al., 1997 Biochimie 79:95-99); (iv) repaglinide, which enhances insulin secretion through interaction with ATP-dependent potassium channels; and (v) acarbose, which decreases intestinal absorption of carbohydrates. Although currently available drugs for treating type 2 diabetes, such as the sulfonylureas, improve insulin secretion, both basal and insulin stimulated insulin secretion are enhanced by such compounds. Consequently, undesirable chronic hyperinsulinemia, hypoglycemia and/or excessive weight gain may result following treatment with such drugs (Cobb et al., 1998 Ann. Rep. Med. Chem. 33:213-222; Krentz et al., 1994 Drug Safety 11:223-241).
None of the current pharmacological therapies corrects the underlying biochemical defect in type 2 DM. Neither do any of these currently available treatments improve all of the physiological abnormalities in type 2 DM such as impaired insulin secretion, insulin resistance and/or excessive hepatic glucose output. In addition, treatment failures are common with these agents, such that multi-drug therapy is frequently necessary.
Mitochondria are organelles that are the main energy source in cells of higher organisms. These organelles provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes, including metabolic energy production, aerobic respiration and intracellular calcium regulation. For example, mitochondria are the site of electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which also underlies a central mitochondrial role in intracellular calcium homeostasis. These processes require the maintenance of a mitochondrial membrane electrochemical potential, and defects in such membrane potential can result in a variety of disorders.
Mitochondria contain an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes. The subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix (for review, see, e.g., Ermster et al., J. Cell Biol. 91:227s, 1981). While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (greater than about 10 kD) molecules.
Four of the five multisubunit protein complexes (Complexes I, III, IV and V) that mediate ETC activity are localized to the inner mitochondrial membrane. The remaining ETC complex (Complex II) is situated in the matrix. In at least three distinct chemical reactions known to take place within the ETC, protons are moved from the mitochondrial matrix, across the inner membrane, to the intermembrane space. This disequilibrium of charged species creates an electrochemical membrane potential of approximately 220 mV referred to as the “proton motive force” (PMF). The PMF, which is often represented by the notation Δp, corresponds to the sum of the electric potential (ΔΨm) and the pH differential (ΔpH) across the inner membrane according to the equationΔp=ΔΨm−ZΔpHwherein Z stands for −2.303 RT/F. The value of Z is −59 at 25° C. when Δp and ΔΨm are expressed in mV and ΔpH is expressed in pH units (see, e.g., Emster et al., J. Cell Biol. 91:227s, 1981, and references cited therein).
ΔΨm provides the energy for phosphorylation of adenosine diphosphate (ADP) to yield ATP by ETC Complex V, a process that is coupled stoichiometrically with transport of a proton into the matrix. ΔΨm is also the driving force for the influx of cytosolic Ca2+ into the mitochondrion. Normal alterations of intramitochondrial Ca2+ are associated with normal metabolic regulation (Dykens, 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 29-55; Radi et al., 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 57-89; Gunter and Pfeiffer, 1991, Am. J. Physiol. 27: C755; Gunter et al., Am. J. Physiol. 267:313, 1994). For example, fluctuating levels of mitochondrial free Ca2+ may be responsible for regulating oxidative metabolism in response to increased ATP utilization, via allosteric regulation of enzymes (reviewed by Crompton and Andreeva, Basic Res. Cardiol. 88:513-523, 1993), and the glycerophosphate shuttle (Gunter and Gunter, J. Bioenerg. Biomembr. 26:471, 1994).
Normal mitochondrial function includes regulation of cytosolic free calcium levels by sequestration of excess Ca2+ within the mitochondrial matrix, including transiently elevated cytosolic free calcium that results from physiologic biological signal transduction. Depending on cell type, cytosolic Ca2+ concentration is typically 50-100 nM. In normally functioning cells, when Ca2+ levels reach 200-300 nM, mitochondria begin to accumulate Ca2+ as a function of the equilibrium between influx via a Ca2+ uniporter in the inner mitochondrial membrane and Ca2+ efflux via both Na+ dependent and Na+ independent calcium carriers, including notably the MCA. The low affinity of this rapid uniporter mechanism suggests that the primary uniporter function may be to lower cytosolic Ca2+ in response to elevation of cytosolic free calcium levels, which may result from calcium influx across the plasma membrane that occurs as part of a biological signal transduction mechanism (Gunter and Gunter, J. Bioenerg. Biomembr. 26:471, 1994; Gunter et al., Am. J. Physiol. 267:313, 1994). In certain instances, for example in pancreatic beta cells, physiologic rises in cytoplasmic calcium occur in response to glucose (or other secretagogues) and lead to calcium uptake by mitochondria, stimulating increased ATP synthesis. Similarly, the primary calcium antiporter (e.g., MCA) function may be to lower mitochondrial Ca2+ concentrations in response to mitochondrial Ca2+ influxes, such as may result from glucose stimulation of a glucose-sensitive cell, and which produce transient increases in oxidative ATP synthesis. Thus, mitochdndrially regulated calcium cycling between, inter alia, cytosolic and mitochondrial compartments may provide an opportunity for manipulation of intracellular ATP levels (e.g., Cox and Matlib, 1993 Trends Pharmacol. Sci. 14:408-413; Matlib et al., 1983 Eur. J. Pharmacol. 89:327; Matlib 1985 J. Pharmacol. Exp. Therap. 233:376; Matlib et al. 1983 Life Sci. 32:2837).
In view of the significance of mitochondrial regulation of intracellular calcium and the relationship of this mitochondrial activity to diabetes, which includes any of a wide range of disease states characterized by inappropriate and sustained hyperglycemia, there is clearly a need for agents to control mitochondrial calcium homeostasis. To provide improved therapies for diabetes, agents that alter mitochondrial calcium cycling between intramitochondrial and extramitochondrial subcellular compartments would be beneficial. Further, there is a need for improved therapeutics that are targeted to correct biochemical and/or metabolic defects responsible for, or associated with, type 2 DM, regardless of whether such a defect underlying altered mitochondrial function may have mitochondrial or extramitochondrial origins.
Accordingly, there is a need in the art agents that modulate mitochondrial calcium/sodium antiporter function and are thus useful for treating diabetes, type 2 DM, by enhancing insulin secretion. There is also a need for pharmaceutical compositions containing such agents, as well as for methods relating to use thereof. The present invention fulfills these needs, and provides further related advantages.